Artesunate Suppresses Oxidative and Inflammatory Processes by Activating Nrf2 and ROS‑Dependent P38 MAPK and Protects Against Cerebral Ischemia‑Reperfusion Injury

MOLECULAR MEDICINE REPORTS 17: 6639-6646, 2018

Artesunate suppresses oxidative and inflammatory processes by activating Nrf2 and ROS‑dependent p38 MAPK and protects against cerebral ischemia‑reperfusion injury

HUI LU1, BINCHENG WANG2, NINGNING CUI1 and YANCHUN ZHANG1

1Department of Neurology, Cangzhou Central Hospital, Cangzhou, Hebei 060000; 2Department of Neurology, Xuan Wu Hospital, Beijing 100053, P.R. China

Received June 14, 2016; Accepted April 25, 2017

DOI: 10.3892/mmr.2018.8666

Abstract. Artesunate is a semi‑synthetic derivative of arte- Introduction misinin that is used in the treatment of patients with malaria. Artesunate has also been reported to exert immune‑regulatory, Cerebrovascular diseases are common conditions caused antitumor, hepatoprotective, anti‑inflammatory and smooth by impairments in the oxygen supply to the brain, among muscle relaxing functions. The present study aimed to inves- which stroke is characterized by a high incidence rate, and is tigate the putative protective effects of artesunate against one of the most common causes of morbidity and mortality cerebral ischemia/reperfusion injury (CIRI), and to elucidate worldwide (1,2). Comorbidities associated with stroke include the molecular mechanisms underlying its effects. A CIRI visual impairments, loss of speech, paralysis and mental mouse model was created via middle cerebral artery occlu- disorders (3). Stroke severely degrades the health and quality sion for 2 h, followed by 22 h of reperfusion. Mice were of life of surviving patients, and it poses a heavy economic and treated with 10‑40 mg/kg artesunate. The present results psychological burden for the patients and their families, and a demonstrated that treatment with artesunate significantly major health concern for society (3). reduced the cerebral infarct volume and potentiated the Stroke is an acute disorder caused by brain hypoxia or recovery of neurological function in CIRI mice. Oxidative ischemia, as a result of blood vessel blockage or cerebral stress and inflammation markers were revealed to be signifi- blood vessel rupture and subsequent hemorrhage (4). Strokes cantly downregulated following treatment with artesunate may thus be divided into ischemic strokes and hemorrhagic in CIRI mice. Furthermore, artesunate was demonstrated to strokes, with ischemic strokes accounting for ~87% of all activate nuclear factor erythroid 2‑related factor 2 (Nrf2), stroke cases. Cerebral ischemia can rapidly cause the necrosis inhibit caspase‑3 activity, reduce the apoptosis regulator of cerebral tissue in the ischemic nucleus, followed by tardive BAX/apoptosis regulator Bcl‑2 expression ratio and suppress neuronal death in ischemic and surrounding tissues, which the phosphorylation of the mitogen‑activated protein kinase ultimately results in brain damage (5). The primary challenge (MAPK) p38 in CIRI mice. In conclusion, the present find- to the treatment of ischemic stroke is the short time window ings suggested that artesunate may exert protective effects for successful intervention; as a result, a large number of against CIRI through the suppression of oxidative and inflam- stroke victims fail to be treated in a timely and effective matory processes, via activating Nrf2 and downregulating manner, with severe consequences to the restoration of brain ROS‑dependent p38 MAPK in mice. function (6). Disturbances in energy homeostasis are among the main causes of cerebral ischemia/reperfusion injury (CIRI) (7). Oxidative stress is involved in CIRI, as the dysregulation of mitochondrial oxidative phosphorylation leads to a decrease in ATP production and an increase in reactive oxygen species (ROS) generation (7). In addition to the decrease in ATP levels and the increased production of free radicals, which can directly damage cellular components, adaptive regula- Correspondence to: Dr Hui Lu, Department of Neurology, Cangzhou Central Hospital, Thomas Regency East 3‑1‑2403, tion mechanisms of CIRI also participate in the molecular Cangzhou, Hebei 060000, P.R. China mechanisms underlying the pathology of the disorder (8). E‑mail: [email protected] The area of the ischemic event is composed of a necrotic area in the infarct focus, and a surrounding ischemic Key words: artesunate, cerebral ischemia‑reperfusion injury, penumbra (9). Cells in the center of the infarction are largely oxidation, inflammation, nuclear factor erythroid 2‑related factor 2, necrotic, whereas the ischemic penumbra is mainly charac- mitogen‑activated protein kinase p38 terized by cellular apoptosis; however, penumbral cells may be salvaged following reperfusion (9). Cellular apoptosis is 6640 LU et al: ARTESUNATE PROTECTS AGAINST CEREBRAL ISCHEMIA-REPERFUSION INJURY a physiological process that can be activated by internal and external factors, and is regulated by several genes, enzymes and signal transduction pathways (9). The mitogen‑activated protein kinase (MAPK) cascade communicates signals from the cell surface to the nucleus and is involved in numerous physiological cellular processes, including adaptation, proliferation, differentiation, survival and apoptosis (10). Therefore, the implication of the MAPK pathway in the molecular mechanisms of CIRI has garnered considerable attention. p38 MAPK is a critical member of the MAPK family, which can be phosphorylated in response to various extracel- lular stimuli, thus activating the MAPK signaling pathway (11). P38 MAPK has been implicated in the regulation of inflammatory responses, and has been associated with cellular Figure 1. Chemical structure of artesunate. differentiation, cell cycle progression and apoptosis (12). During CIRI, p38/MAPK signaling has been reported to be activated, as p38 MAPK has been demonstrated to translocate were subjected to middle cerebral artery occlusion for 2 h, from the cytoplasm into the nucleus, activate downstream followed by 22 h reperfusion, and treated with normal saline kinases and transcription factors, and modulate the expres- for 7 days; CIRI + low artesunate group (n=6), CIRI mice were sion of apoptosis‑associated genes, thus promoting cellular treated with 10 mg/kg/day artesunate (intraperitoneal admin- apoptosis (13). istration, Sigma‑Aldrich; Merck KGaA, Darmstadt, Germany) The extracts of the Chinese medicinal herb for 7 days; CIRI + medium artesunate group (n=6), CIRI mice Artemisia carvifolia have demonstrated diverse phar- were treated with 20 mg/kg/day artesunate (intraperitoneal macological properties, including immune‑regulatory, administration) for 7 days; and CIRI + high artesunate group anti‑inflammatory, antitumor and anti‑angiogenetic (n=6), CIRI mice were treated with 40 mg/kg/day artesunate effects (14). Artesunate (dihydroartemisinin 1,2 α succinic (intraperitoneal administration) for 7 days. acid monoester) is a semi‑synthetic sesquiterpene lactonic derivative of the antimalarial drug artemisinin, which is one Establishment of CIRI mouse model. Mice were anesthetized of the main active ingredients of Artemisia carvifolia (15). with 35 mg/kg sodium pentobarbital. A midline incision Artesunate is a novel antimalarial drug, characterized by was conducted, and the right common, external and internal high efficacy, fast action, low toxicity and low tolerance (15). carotid arteries were exposed. The right common and external In addition, artesunate has exhibited anti‑tumor activity carotid arteries were ligated using 6‑0 sutures, and the internal accompanied by low toxicity and has been used success- carotid artery was subsequently occluded using a clamp. A fully for the treatment of patients with metastatic melanoma silicone‑coated 6‑0 nylon monofilament was inserted into in clinical practice (15). Previous studies have reported that the internal carotid artery from a small incision on the right artesunate increased the intracellular production of oxygen common carotid artery, until the tip blocked the origin of the free radicals, and intervened in nuclear factor (NF)‑κΒ‑ and middle cerebral artery. The surgical incisions were closed phosphatidylinositol‑4,5‑bisphosphate 3‑kinase/Akt‑mediated with 6‑0 sutures, mice were maintained at 37˚C for 2 h, signaling pathways, thus causing DNA damage, interfering and the clamp was subsequently removed for reperfusion. with cell cycle regulation, and preventing metastasis and Subsequently, mice were treated with artesunate. multidrug resistance (15‑17). Therefore, the present study investigated the effects of artesunate during CIRI and the Neurological evaluation. Following treatment with arte- possible molecular mechanisms underlying its actions. sunate, the neurological function of mice was examined as previously described (18). The following grading system Materials and methods was used for evaluation: 0, No neural function deficit; 1, left forepaw weakness; 2, constantly turning left; 3, mouse falling Animals and experimental protocol. The experimental proce- to the contralateral side; 4, mouse unable to walk spontaneously dures used in the present study were reviewed and approved or comatose. by the Animal Experiment Ethics Committee of Cangzhou Central Hospital (Cangzhou, China). Male adult Kunming Histological examination. Following treatment with artesu- mice (weight, 21‑23 g; age, 6‑7 weeks; n=30) were purchased nate, the brains were isolated and sectioned into 4 mm coronal from the Experimental Animal Center of Cangzhou Central slices. To evaluate cerebral infarct volume, the brain tissue Hospital and were allowed to acclimate to lab conditions for sections were incubated with 1% 2,3,5‑triphenyl‑tetrazolium 1 week prior to the commencement of experiments. Mice chloride for 30 min at 37˚C in the dark and then fixed with were housed at a temperature of 21‑23˚C and 55‑60% relative 10% formalin at room temperature for 24 h. Photographs of humidity, under a 12‑h light/dark cycle, with free access to stained brain tissue samples were captured using a digital food and water. camera (Nikon Corporation, Tokyo, Japan) and analyzed using Mice were randomly assigned to the following 5 groups: ImageJ software (version 3.1; National Institutes of Health, Sham‑operated group (n=6); CIRI model group (n=6), mice Bethesda, MD, USA). For histopathological examination, MOLECULAR MEDICINE REPORTS 17: 6639-6646, 2018 6641

Figure 2. Treatment with artesunate reduces cerebral infarct volume and protects neurological function in CIRI mice. (A) Cerebral infarct volumes were significantly reduced in CIRI mice following treatment with artesunate. (B) Treatment with artesunate prevented CIRI‑associated deficits in neurological function. Sham, sham‑operated group; CIRI, CIRI model group; Low, CIRI + 10 mg/kg artesunate for 7 days group; Medium, CIRI + 20 mg/kg artesunate for 7 days group; High, CIRI + 40 mg/kg artesunate for 7 days group. ##P<0.01 vs. Sham; **P<0.01 vs. CIRI. CIRI, cerebral ischemia/reperfusion injury.

hippocampi were isolated, fixed in 10% formalin at room (Bax; 1:500; cat. no. sc‑6236; Santa Cruz Biotechnology, Inc.), temperature for 24 h, embedded in paraffin and sliced into anti‑phosphorylated (p)‑p38 MAPK (1:500; cat. no. sc‑7975‑R; 4‑µm sections. Subsequently, tissue sections were deparaf- Santa Cruz Biotechnology, Inc.) and anti‑GAPDH (1:1,000; finized and stained with hematoxylin and eosin (H&E) at room cat. no. sc‑367714; Santa Cruz Biotechnology, Inc.). Membranes temperature for 10‑15 min and observed with a fluorescence were washed with TBS containing Tween‑20 (0.1%) and microscope (magnification, x10; Zeiss GmbH, Jena, Germany). then incubated with horseradish peroxidase‑conjugated anti‑rabbit IgG (H+L), Biotinylated Antibody (1:5,000; cat. Evaluation of proinflammatory factors, oxidative stress and no. 14708; Cell Signaling Technology, Inc.) at 37˚C for 2 h. caspase‑3 activity. Blood samples were collected and the Protein bands were visualized using an enhanced chemilumi- supernatants were isolated after 2,000 x g for 10 min at 4˚C nescence system (Beyotime Institute of Biotechnology) and to measure the activity of interleukin (IL)‑1β (cat. no. H002), blots were semi‑quantified using ImageJ software version 3.1 IL‑6 (cat. no. H007), IL‑10 (cat. no. H009), tumor necrosis (National Institutes of Health). factor (TNF)‑α (cat. no. H052), glutathione (GSH; cat. no. A006‑2) and superoxide dismutase (SOD; cat. no. A001‑2) Statistical analysis. All data are expressed as the using ELISA kits (Nanjing Jiancheng Bioengineering Institute, mean ± standard deviation (n=3). Statistical analysis was Nanjing, China). The absorbance of the samples was measured performed with SPSS 17.0 software (SPSS, Inc., Chicago, IL, at 450 nm using a Model 680 microplate reader (Bio‑Rad USA). The statistical significance of the differences between Laboratories, Inc., Hercules, CA, USA). ROS production groups was assessed using one‑way analysis of variance with was evaluated using a Reactive Oxygen Species Assay kit Tukey post hoc test. P<0.05 was considered to indicate a (Beyotime Institute of Biotechnology, Haimen, China) with statistically significant difference. a Model 680 microplate reader (Bio‑Rad Laboratories, Inc.), using an excitation wavelength of 488 nm and an emission Results wavelength of 525 nm. Caspase‑3 activity was measured using a Caspase‑3 Activity Assay kit (Beyotime Institute of Treatment with artesunate reduces cerebral infarct volume Biotechnology). The absorbance of the samples was measured and protects neurological function in CIRI mice. The at 405 nm using a Model 680 microplate reader (Bio‑Rad chemical structure of artesunate is presented in Fig. 1. The Laboratories, Inc.). effects of artesunate on cerebral infarct volume and its putative protective effects against neurological function impairments Western blot analysis. The hippocampus and cortex of the were investigated in mice following CIRI. As demonstrated in injured hemisphere were isolated and homogenized in cold lysis Fig. 2A, cerebral infarct volumes in mice from the CIRI model buffer containing phenylmethylsulfonyl fluoride (Beyotime group were significantly larger compared with mice from the Institute of Biotechnology) at 4˚C for 30 min. The superna- sham group, and their neurological function was significantly tants were collected following centrifugation 12,000 x g for impaired (Fig. 2B). However, following treatment with 20 10 min at 4˚C and protein concentration was measured using or 40 mg/kg artesunate, the cerebral infarct volume in CIRI a bicinchoninic acid protein assay kit (Beyotime Institute of mice was significantly reduced compared with untreated mice Biotechnology). Equal amounts of extracted protein samples from the CIRI model group (Fig. 2A). In addition, treatment (50 µg) were separated by 10% SDS‑PAGE and transferred with medium and high doses of artesunate was revealed to onto polyvinylidene difluoride membranes. Membranes significantly protect mice from CIRI‑associated neurological were blocked with 5% milk in TBST at room temperature deficits (Fig. 2B). for 1 h and incubated overnight at 4˚C with the following primary antibodies: Anti‑ nuclear factor erythroid 2‑related Treatment with artesunate alleviates cerebral histopatho‑ factor 2 (Nrf2; 1:500; cat. no. sc‑722; Santa Cruz Biotechnology, logical alterations in CIRI mice. In order to investigate the Inc.), anti‑ apoptosis regulator Bcl‑2 (Bcl‑2; 1:500; cat. no. sc‑783; putative protective effects of artesunate against CIRI‑induced Santa Cruz Biotechnology, Inc.), anti‑apoptosis regulator BAX histopathological alterations in cerebral tissue, hippocampi 6642 LU et al: ARTESUNATE PROTECTS AGAINST CEREBRAL ISCHEMIA-REPERFUSION INJURY

Figure 3. Treatment with artesunate alleviates cerebral histopathological alterations in CIRI mice. Hippocampal tissue samples were isolated and histological examination was performed following staining with hematoxylin and eosin (magnification, x10). Sham, sham‑operated group; CIRI, CIRI model group; Low, CIRI + 10 mg/kg artesunate for 7 days group; Medium, CIRI + 20 mg/kg artesunate for 7 days group; High, CIRI + 40 mg/kg artesunate for 7 days group. CIRI, cerebral ischemia/reperfusion injury.

Figure 4. Treatment with artesunate protects endogenous antioxidant activity in CIRI mice. Treatment with artesunate counteracted the CIRI‑associated suppression of (A) SOD and (B) GSH activity. Sham, sham‑operated group; CIRI, CIRI model group; Low, CIRI + 10 mg/kg artesunate for 7 days group; Medium, CIRI + 20 mg/kg artesunate for 7 days group; High, CIRI + 40 mg/kg artesunate for 7 days group. ##P<0.01 vs. Sham; **P<0.01 vs. CIRI. CIRI, cerebral ischemia/reperfusion injury; SOD, superoxide dismutase; GSH, glutathione.

were isolated and stained with H&E. As presented in Fig. 3, mice from the CIRI model group exhibited severe histological damage compared with mice from the sham group. Treatment with 20 or 40 mg/kg artesunate was demonstrated to markedly attenuate the CIRI‑associated histopathological alterations in hippocampal tissue compared with tissue samples from untreated CIRI mice (Fig. 3). Figure 5. Treatment with artesunate suppresses ROS production in CIRI Treatment with artesunate protects endogenous antioxidant mice. ROS production was significantly enhanced during CIRI, whereas it was significantly downregulated following treatment with artesunate. Sham, activity in CIRI mice. In order to investigate the antioxidative sham‑operated group; CIRI, CIRI model group; Low, CIRI + 10 mg/kg arte- effects of artesunate during CIRI, the activity of the endogenous sunate for 7 days group; Medium, CIRI + 20 mg/kg artesunate for 7 days antioxidants SOD and GSH were assessed using ELISA kits. group; High, CIRI + 40 mg/kg artesunate for 7 days group. ##P<0.01 vs. Sham; ** Mice from the CIRI model group exhibited significantly P<0.01 vs. CIRI. CIRI, cerebral ischemia/reperfusion injury; ROS, reactive oxygen species. reduced SOD and GSH activity compared with mice from the sham group (Fig. 4A and B). Notably, treatment with 20 or 40 mg/kg artesunate was revealed to restore the activity of SOD and GSH compared with untreated CIRI mice (Fig. 4A and B). IL‑10. In CIRI mice, the activity of TNF‑α, IL‑1β and IL‑6 was significantly enhanced, whereas the activity of IL‑10 was Treatment with artesunate suppresses ROS production significantly suppressed compared with in sham‑operated in CIRI mice. In order to further investigate the effects of mice (Fig. 6A‑D). Following treatment with 20 or 40 mg/kg artesunate on oxidative stress during CIRI, ROS production artesunate, TNF‑α, IL‑1β and IL‑6 activity was significantly was assessed. The present results demonstrated that ROS reduced, whereas the activity of IL‑10 was significantly production was significantly potentiated in CIRI mice (Fig. 5). enhanced compared with untreated mice from the CIRI model However, following treatment with 20 or 40 mg/kg artesunate, group (Fig. 6A‑D). ROS production was significantly suppressed compared with untreated mice from the CIRI model group (Fig. 5). Treatment with artesunate prevents caspase‑3 activation in CIRI mice. In order to investigate the molecular mechanisms Treatment with artesunate suppresses inflammatory responses underlying the protective effects of artesunate during CIRI, in CIRI mice. In order to evaluate the putative anti‑inflammatory the activity of the proapoptotic caspase‑3 was assessed. properties of artesunate during CIRI, ELISA kits were used As revealed in Fig. 7, caspase‑3 activity was significantly to assess the activity of the proinflammatory cytokines IL‑1β, increased in mice from the CIRI model group compared IL‑6 and TNF‑α, and of the anti‑inflammatory cytokine with the sham‑operated mice. Notably, following treatment MOLECULAR MEDICINE REPORTS 17: 6639-6646, 2018 6643

Figure 6. Treatment with artesunate suppresses inflammatory responses in CIRI mice. Following treatment with artesunate the activity of (A) TNF‑α, (B) IL‑1β and (C) IL‑6 was inhibited, whereas the activity of (D) IL‑10 was enhanced. Sham, sham‑operated group; CIRI, CIRI model group; Low, CIRI + 10 mg/kg artesunate for 7 days group; Medium, CIRI + 20 mg/kg artesunate for 7 days group; High, CIRI + 40 mg/kg artesunate for 7 days group. ##P<0.01 vs. Sham; **P<0.01 vs. CIRI. CIRI, cerebral ischemia/reperfusion injury; TNF, tumor necrosis factor; IL, interleukin.

proapoptotic factor Bax and the antiapoptotic factor Bcl‑2 were assessed using western blot analysis (Fig. 8A). The present results demonstrated that the Bax/Bcl‑2 protein expression ratio was significantly increased in CIRI mice compared with in sham‑operated mice (Fig. 8C). Following treatment with artesunate (20 or 40 mg/kg), the Bax/Bcl‑2 protein expression ratio was significantly reduced compared with untreated mice Figure 7. Treatment with artesunate prevents caspase‑3 activation in CIRI from the CIRI model group (Fig. 8C). mice. Caspase‑3 activity was significantly upregulated in CIRI mice, whereas it was significantly suppressed following treatment with artesunate. Sham, sham‑operated group; CIRI, CIRI model group; Low, CIRI + 10 mg/kg Treatment with artesunate suppresses p38 MAPK phosphory‑ artesunate for 7 days group; Medium, CIRI + 20 mg/kg artesunate for 7 days lation in CIRI mice. In order to investigate the involvement group; High, CIRI + 40 mg/kg artesunate for 7 days group. ##P<0.01 vs. Sham; of the MAPK signaling pathway in the effects of artesunate ** P<0.01 vs. CIRI. CIRI, cerebral ischemia/reperfusion injury. during CIRI, the protein expression levels of p‑p38 MAPK were assessed using western blot analysis (Fig. 8A). The present results revealed that the protein expression levels of p‑p38 with artesunate, the activation of caspase‑3 was significantly MAPK were significantly upregulated in CIRI mice compared suppressed compared with untreated CIRI mice (Fig. 7). with in mice from the sham group (Fig. 8D). Following treatment with 20 or 40 mg/kg artesunate, p‑p38 protein expression Treatment with artesunate restores Nrf2 protein expression in levels in CIRI mice were significantly reduced compared with CIRI mice. To further investigate the protective mechanisms untreated mice from the CIRI model group (Fig. 8D). implicated in the effects of artesunate during CIRI, western blot analysis was used to assess the protein expression levels Discussion of the transcription factor Nrf2 (Fig. 8A). As demonstrated in Fig. 8B, Nrf2 protein expression was significantly downregu- Cerebrovascular disease is also known as cerebrovascular lated in CIRI mice compared with mice from the sham group. accident or stroke, and is a common disease of the cerebral Conversely, treatment with 20 or 40 mg/kg artesunate was circulation with a high incidence rate (19). According to the revealed to significantly upregulate Nrf2 protein expression third national survey of mortality causes released by the in CIRI mice compared with untreated mice from the CIRI Ministry of Health in 2012, 136.64 out of 100,000 patients with model group (Fig. 8B). stroke succumbed in China, raising the mortality of strokes to more than that of malignant tumors (20). In China, stroke Treatment with artesunate restores the Bax/Bcl‑2 expression has become the disease with the highest mortality rate (21). ratio in CIRI mice. To further explore the anti‑apoptotic effects Thrombolysis, angioplasty, rehabilitation and other therapies, of artesunate during CIRI, the protein expression levels of the as well as early intervention, can restore cerebral blood supply 6644 LU et al: ARTESUNATE PROTECTS AGAINST CEREBRAL ISCHEMIA-REPERFUSION INJURY

Figure 8. Treatment with artesunate restores Nrf2 protein expression and the Bax/Bcl‑2 ratio, and suppresses p38 MAPK phosphorylation in CIRI mice. (A) Protein expression was detected using western blot analysis. (B) Blots were semi‑quantified and statistical analysis demonstrated that treatment of CIRI mice with artesunate prevented the CIRI‑induced downregulation of Nrf2 protein expression. (C) Statistical analysis demonstrated that treatment of CIRI mice with artesunate prevented the CIRI‑induced increase in the Bax/Bcl‑2 protein expression ratio. (D) Statistical analysis demonstrated that treatment of CIRI mice with artesunate prevented the CIRI‑induced upregulation of p‑p38 MAPK protein expression. Sham, sham‑operated group; CIRI, CIRI model group; Low, CIRI + 10 mg/kg artesunate for 7 days group; Medium, CIRI + 20 mg/kg artesunate for 7 days group; High, CIRI + 40 mg/kg artesunate for 7 days group. ##P<0.01 vs. Sham; **P<0.01 vs. CIRI. Nrf2, nuclear factor erythroid 2‑related factor 2; CIRI, cerebral ischemia/reperfusion injury; Bax, apoptosis regulator Bax; Bcl‑2, apoptosis regulator Bcl‑2; p, phosphorylated; MAPK, mitogen‑activated protein kinase. in patients with stroke and alleviate neuronal damage, thus peroxide free radicals, resulting in membrane lipid peroxida- improving the survival rate of stroke victims (22). However, tion and cell damage (27). Therefore, during CIRI, oxidative following the restoration of the blood supply to ischemic stress is among the main causes of neuronal injury. In the tissues, the structure and function of cells undergo further present study, artesunate was revealed to significantly inhibit damage caused by reperfusion, thus resulting in the develop- ROS production in mice with CIRI. Cheng et al (14) suggested ment of CIRI (19). The results of the present study suggested that artesunate may induce endothelial cell apoptosis through that treatment with artesunate may prevent CIRI‑induced an ROS‑dependent p38 MAPK/mitochondrial pathway. neurological impairments, reduce the cerebral infarct volume Conversely, the present findings suggested that the regulation and attenuate CIRI‑associated histological damage in mice. of ROS production may be implicated in the protective effects Oxidative stress results from an imbalance between oxida- of artesunate during CIRI. tive and antioxidative processes, and may cause severe cell Mitochondria are important organelles during the execu- damage (8). An increase in the production of reactive oxygen tion of apoptotic signals in neurons. Cerebral ischemia and and nitrogen species, and other free radicals, is the main reperfusion enhance the mitochondrial production of ROS, cause of oxidative stress (23). ROS can interfere with gene which, through the activation of the proapoptotic protein Bax, expression regulation, thus altering the expression of signaling trigger the mitochondrial release of cytochrome c, which binds proteins, and affecting intracellular signaling cascades (24). with apoptotic protease activating factor 1 and caspase‑9 to Physiological ROS generation is critical for the maintenance form the apoptosome, resulting in the activation of caspase‑3 of normal cellular functions; however, under pathological and other caspases, including caspase‑2, ‑6, ‑8 and ‑10 (28). conditions, including cerebral ischemia, aberrant ROS produc- Activated caspase‑3 may subsequently cleave DNA repair tion can result in the oxidation of cellular components, thus enzymes, thus inhibiting the repair of ischemia‑induced DNA causing cell damage (25). In the present study, artesunate was damage, which results in cellular apoptosis (29). Oxidative demonstrated to significantly attenuate CIRI‑induced impair- stress and increased ROS production have been implicated ments in the activity of the antioxidative enzymes SOD and in the development of CIRI (30). Under physiological condi- GSH in mice. tions, low levels of ROS serve as signaling molecules in Neurons are characterized by high metabolic activity and various cellular processes; however, in pathological states, increased oxygen consumption. In addition, their relatively low when the endogenous antioxidative mechanisms are unable to endogenous antioxidant contents make neurons particularly counteract excessive ROS production, high levels of ROS can sensitive to oxidative stress (26). Furthermore, the brain is oxidize lipids, proteins, nucleic acids and intracellular compo- rich in lipids, which can react with ROS to generate hydrogen nents, thus causing cell damage (31). Direct or indirect protein MOLECULAR MEDICINE REPORTS 17: 6639-6646, 2018 6645 oxidation by ROS can alter their structure, increase their 5. Shan J, Sun L, Wang D and Li X: Comparison of the neuroprotective effects and recovery profiles of isoflurane, hydrophobicity and interfere with protein‑protein interactions, sevoflurane and desflurane as neurosurgical pre‑conditioning leading to the formation of protein aggregates, and causing on ischemia/reperfusion cerebral injury. Int J Clin Exp Pathol 8: cell damage (32). In the present study, artesunate was revealed 2001‑2009, 2015. 6. Jiang F, Yang J, Zhang L, Li R, Zhuo L, Sun L and Zhao Q: to significantly suppress the activity of the proapoptotic Rosuvastatin reduces ischemia‑reperfusion injury in patients caspase‑3, reduce the Bax/Bcl‑2 protein expression ratio and with acute coronary syndrome treated with percutaneous coro- upregulate the expression of the antiapoptotic transcription nary intervention. 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